Greatly Enhanced CTC Culture Enabled by Capturing CTC Heterogeneity Using a PEGylated PDMS-Titanium-Gold Electromicrofluidic Device with Glutathione-Controlled Gentle Cell Release.

The circulating tumor cells (CTCs, the root cause of cancer metastasis and poor cancer prognosis) are very difficult to culture for scale-up in vitro, which has hampered their use in cancer research/prognosis and patient-specific therapeutic development. Herein, we report a robust electromicrofluidic chip for not only efficient capture of heterogeneous (EpCAM+ and CD44+) CTCs with high purity but also glutathione-controlled gentle release of the CTCs with high efficiency and viability. This is enabled by coating the polydimethylsiloxane (PDMS) surface in the device with a 10 nm gold layer through a 4 nm titanium coupling layer, for convenient PEGylation and linkage of capture antibodies via the thiol-gold chemistry. Surprisingly, the percentage of EpCAM+ mammary CTCs can be as low as ∼35% (∼70% on average), showing that the commonly used approach of capturing CTCs with EpCAM alone may miss many EpCAM- CTCs. Furthermore, the CD44+ CTCs can be cultured to form 3D spheroids efficiently for scale-up. In contrast, the CTCs captured with EpCAM alone are poor in proliferation in vitro, consistent with the literature. By capture of the CTC heterogeneity, the percentage of stage IV patients whose CTCs can be successfully cultured/scaled up is improved from 12.5% to 68.8%. These findings demonstrate that the common practice of CTC capture with EpCAM alone misses the CTC heterogeneity including the critical CD44+ CTCs. This study may be valuable to the procurement and scale-up of heterogeneous CTCs, to facilitate the understanding of cancer metastasis and the development of cancer metastasis-targeted personalized cancer therapies conveniently via the minimally invasive liquid/blood biopsy.

Association of Serum Progranulin Levels With Disease Progression, Therapy Response and Survival in Patients With Metastatic Breast Cancer.

BACKGROUND: Progranulin (GP88) is a critical player in breast tumorigenesis. GP88 tumor expression is associated with increased recurrence and mortality, whereas GP88 circulating levels are elevated in patients with breast cancer compared with healthy individuals. We examined here the correlation between serum GP88 levels in patients with metastatic breast cancer (MBC) with overall survival and disease status determined as response to therapy or progression of disease. PATIENTS AND METHODS: An institutional review board (IRB)-approved study prospectively enrolled 101 patients with MBC at the University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center. GP88 serum levels were correlated with patients' disease status determined by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 criteria and survival outcomes by Kaplan-Meier analysis and log rank statistics. RESULTS: Patients' survival was stratified by serum GP88 level. Patients with serum GP88 < 55 ng/mL had a 4-fold increased survival compared with patients with GP88 > 55 ng/mL. Examination of GP88 serum levels in association with disease status showed a statistically significant association between serum GP88 levels and disease progression or response to therapy while CA15-3 level was only associated to progression. CONCLUSION: The association of serum GP88 level with survival and disease status suggests the potential of using the serum GP88 test for monitoring disease status in patients with MBC. Measurement of serum GP88 levels in patients with MBC may have clinical value as a cost-effective adjunct to the management of patients with MBC with imaging.

Patient-reported Adherence to Adjuvant Aromatase Inhibitor Therapy Using the Morisky Medication Adherence Scale: An Evaluation of Predictors.

OBJECTIVES: Endocrine therapy is part of standard adjuvant therapy for patients with hormone receptor-positive breast cancer and has been shown to improve recurrence-free and overall survival. However, adherence to endocrine therapy is suboptimal and is difficult to measure. In this study we evaluate the feasibility of using the Morisky Medication Adherence Scale (MMAS) to assess patient adherence to aromatase inhibitor (AI) therapy. METHODS: Patients with stage 1 to 3, hormone receptor-positive breast cancer receiving adjuvant AI therapy were prospectively enrolled on an Institutional Review Board approved protocol. The MMAS questionnaire was administered to each patient and adherence was measured. Information on duration of AI therapy, patient and tumor characteristics, and treatment was collected. A multivariable logit model approach was utilized to evaluate potential barriers to adherence. RESULTS: Between 2011 and 2014, 100 patients were enrolled. The distribution of adherence levels was 13% low, 37% medium, and 50% high. High adherence was reported more frequently in white women (58%), patients with stage 2 and 3 disease (54%), and patients who did not receive chemotherapy (62%). Multivariable analysis demonstrated that higher adherence was more likely in white women compared with African American women (estimated odds ratio=2.8). CONCLUSIONS: Using the MMAS, only 50% of women with stage 1 to 3 breast cancer reported high adherence to AI therapy, consistent with other reports showing suboptimal adherence to adjuvant endocrine therapy. The MMAS allows for the rapid assessment of adherence to oral adjuvant endocrine therapy and is valuable in a busy clinical setting.

Trastuzumab-induced cardiomyopathy: incidence and associated risk factors in an inner-city population.

BACKGROUND: Although it is known that trastuzumab causes cardiotoxicity, its extent and reversibility are still in question. Earlier studies have not evaluated consecutive patients with reproducible nuclear ventriculography. OBJECTIVE: We sought to evaluate the baseline characteristics which predispose patients to increased risk of trastuzumab cardiotoxicity and to determine the natural history of the cardiotoxicity. METHODS AND RESULTS: Left ventricular ejection fraction (LVEF) was measured in 76 women aged 36-73 years who had been treated with trastuzumab at the University of Maryland Greenebaum Cancer Center. LVEF was determined at baseline and then 3, 6, 9, and 12 months after treatment initiation. Cardiotoxicity was defined as ≥ 16% decrease in LVEF or ≥ 10% decrease in LVEF to <50%. There were no differences in comorbidities, earlier treatment, or demographics between patients with and without trastuzumab-induced cardiomyopathy except that African Americans were more likely to develop decreased LVEF (P < .05). Twenty-one patients (28%) met criteria for cardiotoxicity. Four of those patients were continued on trastuzumab and 17 patients had therapy withheld at some point. Only 1 patient developed symptomatic heart failure requiring inpatient hospitalization. LVEF improved in most patients regardless of whether or not trastuzumab was continued. CONCLUSIONS: Decreased LVEF while undergoing trastuzumab therapy occurs frequently and is usually reversible. African Americans had a higher risk of developing decreased LVEF. These findings raise clinically important questions as to whether it is necessary to discontinue trastuzumab for asymptomatic decrease in LVEF and whether African Americans are more predisposed to a decrease in LVEF while receiving trastuzumab. Further studies carefully assessing LVEF should address these hypotheses.

A dual-center randomized controlled double blind trial assessing the effect of acupuncture in reducing musculoskeletal symptoms in breast cancer patients taking aromatase inhibitors.

Up to 50 % of women receiving aromatase inhibitor (AI) complain of AI-associated musculoskeletal symptoms (AIMSS) and 15 % discontinue treatment. We conducted a randomized, sham-controlled trial to evaluate whether acupuncture improves AIMSS and to explore potential mechanisms. Postmenopausal women with early stage breast cancer, experiencing AIMSS were randomized to eight weekly real or sham acupuncture sessions. We evaluated changes in the Health Assessment Questionnaire Disability Index (HAQ-DI) and pain visual analog scale (VAS) following the intervention compared to baseline. Serum estradiol, ß-endorphin, and proinflammatory cytokine concentrations were measured pre and post-intervention. We enrolled 51 women of whom 47 were evaluable, including 23 randomized to real and 24 to sham acupuncture. Baseline characteristics were balanced between groups with the exception of a higher HAQ-DI score in the real acupuncture group (p = 0.047). We did not observe a statistically significant difference in reduction of HAQ-DI (p = 0.30) or VAS (p = 0.31) between the two groups. Following eight weekly treatments, we observed a statistically significant reduction of IL-17 (p ≤ 0.009) in both groups. No significant modulation was seen in estradiol, ß-endorphin, or other proinflammatory cytokine concentrations in either group. We did not observe a significant difference in AIMSS changes between real and sham acupuncture. As sham acupuncture used in this study may not be equivalent to placebo, further studies with a non-acupuncture arm may be required to establish whether acupuncture is beneficial for the treatment of AIMSS.

Increased Circulating Level of the Survival Factor GP88 (Progranulin) in the Serum of Breast Cancer Patients When Compared to Healthy Subjects.

INTRODUCTION: GP88 (PC-Cell Derived Growth Factor, progranulin) is a glycoprotein overexpressed in breast tumors and involved in their proliferation and survival. Since GP88 is secreted, an exploratory study was established to compare serum GP88 level between breast cancer patients (BC) and healthy volunteers (HV). METHODS: An IRB approved prospective study enrolled 189 stage 1-4 BC patients and 18 HV. GP88 serum concentration was determined by immunoassay. RESULTS: Serum GP88 level was 28.7 + 5.8 ng/ml in HV and increased to 40.7 + 16.0 ng/ml (P = 0.007) for stage 1-3 and 45.3 + 23.3 ng/ml (P = 0.0007) for stage 4 BC patients. There was no correlation between the GP88 level and BC characteristics such as age, race, tumor grade, ER, PR and HER-2 expression. CONCLUSION: These data suggest that serial testing of serum GP88 levels may have value as a circulating biomarker for detection, monitoring and follow up of BC.

Computer assisted quantitative immunofluorescence of tumor tissue marker expression and clinical outcome to chemotherapy in advanced breast cancer patients.

Chemotherapy is frequently used in the treatment of advanced breast cancer. The identification of patient-specific tumor characteristics that can improve the ability to predict response to chemotherapy would help optimize advanced breast cancer treatment approaches. Quantitative immunofluorescence (QIF) may be applied to the standardization of protein analysis, resulting in increased sensitivity and reproducibility. In the current pilot study, QIF was used to correlate the expression of beta tubulin III and thymidylate synthase with clinical outcome associated with taxane and capecitabine treatment, respectively. QIF analysis is based on fluorescent dye-labeled monoclonal antibody staining followed by computer-assisted microscopy to measure the expression of molecular markers in tumor samples derived from a retrospective database. The interpretation of the tumor marker expression levels results in classification of breast tumors as sensitive or resistant to a mechanistically related drug. Overall diagnostic accuracy of QIF for taxane based therapy was 88% (CI 75.0 - 95.3) with a positive predictive value of 86% and a negative predictive value of 100%, while diagnostic accuracy QIF for capecitabine therapy was 86% (CI 88.0-96.0) with a positive predictive value of 80% and a negative predictive value of 100%. In this study, QIF showed retrospectively a potential for predictive value when analyzing chemotherapeutic treatments for individual advanced stage breast cancer patients. The predictive power of the QIF for chemotherapy confirms that further studies utilizing larger clinical cohorts are warranted.

The significance of circulating epithelial cells in Breast Cancer patients by a novel negative selection method.

PURPOSE: A negative selection method for the enumeration and characterization of circulating epithelial/cancer cells (CCC) in Breast Cancer (BC) patients is described. This manual procedure yields reproducible results of high sensitivity and selectivity suitable for research laboratories. PATIENTS AND METHODS: We conducted a prospective blood sampling study in 105 women with stage 1-4 BC attending clinics at the University of Maryland Greenebaum Cancer Center to define the prevalence of CCC utilizing our sensitive double gradient centrifugation and magnetic cell sorting CCC detection and enumeration method. CCC were isolated and enumerated from 15 to 20 ml of venous blood drawn before the start of systemic therapy and periodically thereafter for up to 24 months. One or more CCC/sample was considered a positive result. RESULTS: We analyzed 487 samples for the presence of CCC; the median number of samples/patient was 4 (range 1-8). CCC were detected in 56% of patients, 19%-stage 1; 43%-stage 2; 46%-stage 3; 83%-stage 4. The probability of being positive for the presence of CCC is significantly associated with the stage of cancer (P < 0.0001). The frequency of CCC positive patients and samples increased with the advancing stage of disease. Presence of more than 10 CCC/sample was associated with the decreased survival and increased probability of having metastatic disease P = 0.001. CONCLUSIONS: Increasing number of CCC/sample correlates with the adverse outcome and poorer survival (P < 0.0001). Our CCC test based on the negative selection procedure may provide valuable prognostic information.

Clinical and pharmacologic study of tributyrin: an oral butyrate prodrug.

PURPOSE: Butyrate is a small polar compound able to produce terminal differentiation and apoptosis in a variety of in vitro models at levels above 50-100 microM. Previously our group demonstrated that daily oral administration of the prodrug, tributyrin, is able to briefly achieve levels >100 microM. Given in vitro data that differentiating activity requires continuous butyrate exposure, the short t1/2 of the drug and a desire to mimic the effects of an intravenous infusion, we evaluated a three times daily schedule. PATIENTS AND METHODS: Enrolled in this study were 20 patients with advanced solid tumors for whom no other therapy was available, had life expectancy greater than 12 weeks, and normal organ function. They were treated with tributyrin at doses from 150 to 200 mg/kg three times daily. Blood was sampled for pharmacokinetic analysis prior to dosing and at 15 and 30 min and 1, 1.5, 2, 2.5, 3, 3.5 and 4 h thereafter. RESULTS: The patients entered comprised 15 men and 5 women with a median age of 61 years (range 30-74 years). Prior therapy regimens included: chemotherapy (median two prior regimens, range none to five), radiation therapy (one), no prior therapy (one). There was no dose-limiting toxicity. Escalation was halted at the 200 mg/kg three times daily level due to the number of capsules required. A median butyrate concentration of 52 microM was obtained but there was considerable interpatient variability. No objective responses were seen. There were four patients with prolonged disease stabilization ranging from 3 to 23 months; median progression-free survival was 55 days. Two patients with chemotherapy-refractory non-small-cell lung cancer had survived for >1 year at the time of this report without evidence of progression. CONCLUSION: Tributyrin is well tolerated and levels associated with in vitro activity are achieved with three times daily dosing.